Initiation of DNA replication in eukaryotic cells is a highly regulated process that is essential for genome integrity. Failure to copy the genome only once and at the proper time during the cell cycle can lead to elevated mutation rates, chromosome instability, and the development of cancer. It is therefore of paramount importance for human health to understand the mechanism of replication initiation. Replication initiation involves a choreographed assembly of several dynamic multiprotein complexes, which must recognize and unwind DNA at origins of replication, interpret checkpoint signals, and ultimately establish the replication fork. Although the order of recruitment of many initiation factors has been defined, the mechanisms by which they assemble the DNA unwinding/synthesis machinery, or replisome, are unknown. The long-range goals of this research program are to understand the molecular mechanism of Mcm10, a central replication factor responsible for activation of pre-replicative complexes and early steps of DNA synthesis. Mcm10 is the first protein to load chromatin at the onset of S-phase, is required for origin unwinding, loading essential replication factors (e.g., Cdc45, RPA) onto chromatin, and stabilizing the catalytic subunit of DNA polymerase a (pol a). Preliminary results from our laboratory have defined the overall domain architecture of Mcm10 from Xenopus laevis, and have identified the regions that interact with DNA and pol a. In the current proposal, we will use the tools of structural biology and biochemistry to determine the physical basis for Mcm10 interaction with DNA (Aim 1) and pol a (Aim 2). X-ray crystallography and NMR spectroscopy will be used jointly to characterize the structures of Mcm10 domains and their protein/DNA complexes. This information will then be placed into a broader functional context using a structure-based mutagenesis approach, taking advantage of interactions with strategically chosen collaborators. This powerful structure-function analysis of Mcm10 is an essential first step toward a mechanistic understanding of how origin unwinding is coupled to replisome assembly and function. A higher resolution description of key replication factors and their intermolecular interactions will lay the foundation for the possible development of therapeutic agents against cancer and other human disease.